CN101200730B - IL-12 expression carrier as well as eukaryotic cell strain expressed thereby and uses thereof - Google Patents

Abstract

The present invention relates to an IL-12 expression vector and eukaryocyte strain expressed by the vector and application. The present invention transcripts two p35 subunits and one p40 subunit of rhIL-12 to be constructed inside a same carrier; each subunit is processed for expression level regulation by a transcription regulating element, and the efficient expression of rhIL-12 is realized by introducing the regulating elements of Intron and SV40Enhancer which can improve the expression level.

Description

The eucaryotic cell strain of IL-12 expression vector and this vector expression and application

Technical field

The invention belongs to biological technical field, specifically the present invention relates to the structure of interleukin 12 highly effective eukaryon expression carrier, the transfection of mammalian cell, rhIL-12 efficiently expresses preparation and the pharmaceutical application of screening, evaluation and the rhIL-12 of cell strain.

Background technology

(recombinant human interleukin12 is that a kind of molecular weight is the gp of 75KD rhIL-12) to rhIL-12, is to lean on disulfide linkage to link the heterodimer that forms by two P35 of subunit, P40.The ability of antiviral, antitumor and metastases that IL-12 has mainly is that it can activating cytotoxic T-lymphocyte ((Brunda etal. (1993) J.Exp.Med.178,1223-1230; ), and produce IFN-γ relevant (Nastala, C.Journal of Immunology, 1994 with its stimulation; 153:1697-1706; Seder, R., Proc.Natl.Acad.Sci.USA, 1993; 90:10188-10192).The startup factor that present known IL-12 is a kind of cellular immunization preferably can be used as the adjuvant that strengthens cellular immunization, has great application prospect.

Vector expression IL-12 such as adenovirus, retrovirus, Epstein-Barr virus, adeno-associated virus and baculovirus have been utilized both at home and abroad.Because IL-12 is a kind of gp, mammalian cell expression system is optimal selection.In the eukaryotic cell expression system, Chen Weifeng (biological chemistry and biophysics progress .1998; 25:254-258), the mode of employing cotransfection such as Liu Meng is expressed bright (the Hunan Medical University's journal 2003 in sheep east in Chinese hamster ovary celI; 28:338-342) utilize double-promoter control to express two subunits of IL-12, and Chen Shishu (Chinese biological chemistry and molecular biosciences journal, 1999; 15:48-53) then adopt IRES to connect the two subunits of mouse IL-12, in the COS cell, express.More than various expression because the unhomogeneity of transfection efficiency, integration efficiency etc., cause that expression efficiency differs, two subunit expression level difference, two subunit do not match each other etc.The expression level of IL-12 is mainly determined by p35 in the present known normal cell, and the p35 expression level is on the low side generally speaking, causes the expression level of IL-12 to be difficult to increase substantially, and its production and application receive very big restriction.The invention solves this key issue of IL-12.

Summary of the invention

The carrier that the purpose of this invention is to provide a kind of rhIL-12 of expression utilizes this carrier in mammal cell line, to efficiently express rhIL-12 and method and its pharmaceutical application, and described rhIL-12 prepares through genetic engineering technique.

In first aspect, the invention provides the method that a kind of acquisition efficiently expresses the rhIL-12 expression vector.There has been data to show, two subunit p35 of interleukin 12 and p40, expression level is unmatched in mammalian cell, normally p40 subunit expression level is higher, and p35 subunit expression level is lower, causes the expression level of rhIL-12 lower.Do not match, be difficult to screen the problem that obtains high expression level purpose clone usually behind the transfectional cell to two subunit expressions among the existing rhIL-12, the carrier of expressing rhIL-12 is carried out brand-new design: the present invention is structured in p35 subunit transcript and p40 subunit transcript in the carrier.Each subunit is all controlled by the CMV promotor respectively, thereby guarantees that two subunit p35 and p40 obtain effective expression.The present invention improves the expression level of p35 subunit with following two kinds of methods; Promptly before the promotor of p35 subunit, designed the enhancer element of SV40; Between promotor and p35 subunit sequence, Intron element (Buchman, AR.Mol.Cell.Biol.1988 have been introduced again; 8,4395-4405.), form the expression unit of a p35 subunit, further, with two p35 subunit expression units in series in same carrier, to increase the expression level of p35.Between p40 subunit and selection markers (Neo) gene, introduce the IRES sequence,, thereby guaranteeing under the elevated pressures condition that screening obtains required clone more easily with raising resistance screening expression of gene level.Through above design, the people IL-12 expression vector primary structure that is obtained is following: SV40Enhancer-pCMV-Intron-p35-SV40Enhancer-pCMV-Intron-p35-pCMV-p40-IRES-neo.Utilize the strain of above-mentioned carrier cells transfected, be easy to obtain high expression level, mate two subunits of rhIL-12 preferably, and then obtain highly active rhIL-12.

Term " protein ", " albumen ", " polypeptide " used among this paper have identical implication." rhIL-12 " can mutual alternative use with " people IL-12 " among this paper.When expressing albumen of the present invention with genetic engineering means; Difference according to employed host cell in producing; As; Yeast cell, vegetable cell, insect cell, mammalian cell, mammal galactophore reactor drum etc., proteic glycosylation form of the present invention can be inconsistent or inhomogenous.

People IL-12 among the present invention, except that naturally occurring amino acid whose form, also comprise all kinds of mutant forms that its N-terminal has the form of methionine(Met), two subunits with and truncated mutant etc.

Preferably, the aminoacid sequence of two subunits of people IL-12 of the present invention is shown in appendix 1a and appendix 1b.

The carrier that is suitable among the present invention includes but not limited to: the various carriers of in mammalian cell, expressing the carrier (vaccinia virus vector, retroviral vector, adenovirus carrier, adeno-associated virus carrier etc.) of usefulness, in plant, expressing the plant viral vector of usefulness and in mammal galactophore, expressing usefulness.In a preferred embodiment, said expression vector is a mammalian cell expression vector.

Aspect second, expression vector comprises selectable marker gene, and the resistant gene that can in eukaryotic cell, adopt includes but not limited to: neomycin resistance gene, Zeocin resistant gene, dihydrofolate reductase gene and hygromycin gene etc.The pressure screening-gene that the present invention adopted is neomycin resistance gene (a neo gene); For the resistant cell that guarantees to filter out after the transfection can more effective expression IL-12; The present invention is gene constructed under the regulation and control of same promotor with neomycin resistance gene and second subunit p40's of interleukin 12, between p40 subunit and resistance screening mark, introduces the IRES element of regulation and control neo genetic expression simultaneously.A series of technology such as those skilled in the art's DNA recombinant technology capable of using make up the dna sequence dna contain proteins encoded according to the invention, suitable expression vector of transcribing with particular element such as translational control sequence, promotor and selected markers.Above-mentioned carrier can be used to transform, the transfection proper host cell, so that obtain needed target protein.

Aspect the 3rd, the invention provides a kind of host cell, it is characterized in that said cell contains the described carrier in first aspect of the present invention, perhaps said cell transforms or transfection with the described nucleic acid molecule in first aspect of the present invention.Host cell can be vegetable cell, insect cell, mammalian cell etc.Like CHO, COS, MDCK (Pei D.et al.Protein Exp and Purif1998; 13:277-281) cell etc.Host cell promptly constitutes through engineering approaches cell or cell strain after containing the gene order of proteins encoded according to the invention in transfection, can be used for producing required IL-12 albumen.Those skilled in the art can select appropriate carriers, host cell rightly; And how to know with carrier high-efficiency be transfected in the host cell, method therefor includes but not limited to: liposome, coprecipitation of calcium phosphate, electro fusion method and microinjection etc.In a preferred embodiment, adopted the liposome transfection method.

Aspect the 4th, the invention provides preparation said people IL-12 proteic method, it may further comprise the steps: the engineering cell that obtains with third aspect of the present invention, cultivate through ordinary method and to express needed albumen.Separation purification method includes but not limited to: smudge cells (UW, osmotic pressure), and centrifugal, saltout molecular sieve; Ion-exchange chromatography, adsorption chromatography, reverse chromatograms, performance liquid chromatography; Capillary electrophoresis, preparation property isoelectrofocusing etc., these methods all are well-known to those skilled in the art.

Aspect the 5th, the invention provides the medicinal application of said IL-12, use as the cellular immunization adjuvant like people IL-12 the present invention's preparation.

Aspect the 6th, the invention provides the application of the pharmaceutical composition of said IL-12, can be used to treat hepatitis B like people IL-12 with the present invention's preparation; Pharmaceutical composition of the present invention comprises other therapeutical agent, and like this, according to the method for the invention, the pharmaceutical composition that includes people IL-12 of the present invention can be used as the part of combined therapy, uses with other medicament combination.

Accompanying drawing among the present invention is used to explain specific embodiments of the present invention, and is not used in qualification by the scope of the invention that claims defined.

The present invention has quoted open source literature, and these documents are in order more clearly to describe the present invention, and their full text content is all included this paper in and carried out reference, just looks like that repeated description is the same excessively in this article for their full text.

For the ease of understanding, below will the present invention be described in detail through concrete embodiment.What need particularly point out is that these descriptions only are exemplary descriptions, do not constitute limitation of the scope of the invention.According to the argumentation of this specification sheets, many variations of the present invention, change have been obviously all concerning one of ordinary skill in the art.

Description of drawings

Fig. 1 is p35 gene and p40 gene PCR product;

Fig. 2 is a recombinant plasmid pDouble-P35 structural representation;

Fig. 3 is a recombinant plasmid pP40IRESneo structural representation;

Fig. 4 is a recombinant plasmid pP75 structural representation;

Fig. 5 recombinant plasmid pDouble-P35 enzyme is cut evaluation;

Fig. 6 recombinant plasmid pP40IRESneo enzyme is cut evaluation, among the figure: M1.DNA marker DL2000; M2.DNAmarker DL15000; 1.KpnI single endonuclease digestion 2.EcoRI single endonuclease digestion

Fig. 7 recombinant plasmid pP75 enzyme is cut evaluation, among the figure: M1.DNA marker DL2000; M2.DNA markerDL15000; 1.EcoRI single endonuclease digestion; 2.EcoRV single endonuclease digestion

Fig. 8 p2P35-1P40 structural representation;

Fig. 9 IL-12 stoste sex change and non-sex change SDS-PAGE, remarks: 1. sex change IL-12; Molecular weight Marker (116kD, 66.2kD, 45kD, 35kD, 25kD, 18.4kD, 14.4kD); 3. non-sex change IL-12

The molecular weight detection MALDI-TOF-MS figure of Figure 10 rhIL-12;

The Western Blot of Figure 11 IL-12 detects;

The mass spectroscopy peptide figure of Figure 12 IL-12.

Embodiment

The following stated experimental technique does not specify, and all carries out according to the said method in " molecular cloning experiment guide " (2002, Science Press).

Embodiment 1

Design of primers and gene amplification

Design following p35 subunit amplimer and p40 subunit amplimer (entrusting Shanghai to give birth to worker's biotechnology ltd synthesizes); Extract total RNA that test kit (Qiagen Company products) extracts people's cell with RNeasy; And as the template of reverse transcription PCR; Carry out single stage method RT-pcr amplification (Takara company test kit) respectively, obtaining length is the p35 gene product of 684bp and the p40 gene product (see figure 1) of length 1002bp, all consistent with the expection size.

P35 forward primer sequence is (underscore is represented the SalI restriction enzyme site):

5’-TGG GTCGACGCCACCATGTGTCCAGCGCGCAGCCTC-3’

P35 reverse primer sequence is (underscore is represented the SalI restriction enzyme site):

5’-TGG GTCGACTCAGGAAGCATTCAGATAGC-3’

P40 forward primer sequence is (underscore is represented the BamHI restriction enzyme site):

5’-CG GGATCCATGTGTCACCAGCAGTTG-3’

P35 reverse primer sequence is (underscore is represented the BamHI restriction enzyme site):

5’-CG GGATCCTAACTGCAGGGCACAGATG-3’

Embodiment 2

Gene clone

(1) structure of recombinant plasmid pDouble-P35

Carrier pDouble carries double expression boxes.The PCR product of p35 gene is inserted in the pDouble carrier with SalI single endonuclease digestion rear clone, and screening obtains recombinant plasmid and the called after pDouble-P35 (structural representation is seen Fig. 2) that forward inserts.

(2) structure of recombinant plasmid pP40IRESneo

The carrier pIRESneo that carries Neo resistance screening mark is available from U.S. Clontech company.The PCR product of p40 gene is inserted in the pIRESneo carrier with BamHI single endonuclease digestion rear clone, and screening obtains recombinant plasmid and the called after pP40IRESneo (structural representation is seen Fig. 3) that forward inserts.

(3) structure of recombinant plasmid p75

With NruI+XhoI double digestion carrier pP40IRESneo, the expression cassette that carries whole P40-IRES-Neo is transferred to the KpnI single endonuclease digestion and mends in the pDouble-P35 carrier after putting down the recombinant plasmid called after pP75 (structural representation is seen Fig. 4) of acquisition.

Embodiment 3

The evaluation of gene and sequencing

The evaluation of recombinant plasmid pDouble-P35, pP40IRESneo and pP75

(1) with EcoRI and BglII single endonuclease digestion pDouble-P35 is carried out enzyme respectively and cut evaluation; Obtain two bands (EcoRI single endonuclease digestion) and big or small two bands (the BglII enzyme is cut) that are about 1.3kb and 5.4kb that size is about 2.3kb and 4.4kb, result and expection consistent (Fig. 5).

(2) with KpnI and EcoRI single endonuclease digestion pP40IRESneo is carried out enzyme respectively and cut evaluation; Obtain two bands (KpnI single endonuclease digestion) and big or small two bands (EcoRI single endonuclease digestion) that are about 0.8kb and 5.4kb that size is about 1.9kb and 4.3kb, result and expection consistent (Fig. 6).

(3) with EcoRI and EcoRV single endonuclease digestion pP75 is carried out enzyme respectively and cut evaluation; Obtain four bands (the EcoRI enzyme is cut) and big or small three bands (EcoRV single endonuclease digestion) that are about 0.46kb, 0.95kb and 8kb that size is about 0.76kb, 1.04kb, 2.25kb and 5.37kb, result and expection consistent (Fig. 7).

Embodiment 4

Genetic engineering modified and the Construction of eukaryotic of P35

Carry the construction of recombinant plasmid of 2 P35 subunit expression boxes and 1 P40 subunit expression box

(1), reclaims P35 subunit expression box (3285bp), and mend flat through the T4DNA polysaccharase with NruI+KpnI double digestion carrier pDouble-P35 (Fig. 2).

(2), carry out dephosphorylation (9416bp) with the CIP enzyme with NruI single endonuclease digestion carrier pP75 (Fig. 4).

(3) connect 1 and 2, filter out positive colony p2P35-1P40 (Fig. 8) behind the transformed into escherichia coli.

Embodiment 5

The expression level of IL-12 detects behind the recombinant plasmid p2P35-1P40 transient transfection HeLa cell

The HeLa cell inserts six orifice plates, is cultured to 90% individual layer, and recombinant plasmid p2P35-1P40 is with Lipofectamine2000 transfection reagent (Invitrogen Company products) transfection HeLa cell, and the operation by specification carries out, and 3 plasmid transfection holes and 3 control wells are set.After transfection, collect whole supernatant nutrient solutions in 24 hours and add fresh substratum and continue to cultivate, after transfection, also collected the supernatant nutrient solution by as above operating respectively in 48,72 and 96 hours.The expression level of IL-12 detects through ELISA test kit (brilliant U.S. Company products), and average expression amount is seen table 1.

The expression level of IL-12 detects behind the table 1 recombinant plasmid p2P35-1P40 transient transfection HeLa cell

	The expression amount of p2P35-1P40 plasmid transfection hole IL-12	The expression amount of control wells IL-12
			After the transfection 24 hours	22.4ng/ml	<1ng/ml
After the transfection 48 hours	186ng/ml	<1ng/ml
			After the transfection 72 hours	256ng/ml	<1ng/ml
After the transfection 96 hours	63.9ng/ml	<1ng/ml

Embodiment 6

The transfection of Chinese hamster ovary celI and screening

Adopt the Lipofectin of GIBCO/BRL to carry out cell transfecting, the operation by specification carries out.Expression vector p2P35-1P40 is transfection CHO cell behind the PEG purifying.Chinese hamster ovary celI is with 1640 perfect mediums (containing 10% calf serum), 37 ℃ of 5%CO ₂Be cultured to the about 80% remittance sheet of cell.Press the test kit description operation, collect supernatant behind the transfection 72h, add the G418 screening.Cultivate about two weeks, after having obvious clone to form, remove G418.Collect the culturing cell supernatant after a couple of days, carry out the biological and immunocompetent detection of IL_12.Picking active high clone be transferred in 24 orifice plates, transfers in 6 orifice plates and cultivate, and the screening of pressurizeing obtains to express the high clone of IL-12.Therefrom detect the highest active clone, form engineering cell strain.

Embodiment 7

The cultivation of recombinaant CHO cell

Chinese hamster ovary (CHO) but cell both adherent culture also can suspended pattern growth, we progressively carry out the transition to few serum and cultivate from containing the serum adherent culture, so that the floated cultivation of serum-free.The advantage that the continous pouring of the cell biological reactor drum of serum-free is cultivated is the culture condition stable and controllable; The state of being convenient to pair cell is assessed accurately; Strengthen the detection of pair cell external environment, help increasing the secretion of cell target protein, make the purifying of target protein become and carry out easily.I cultivate the serum content that gradually reduces nutrient solution from containing serum in the laboratory, successfully am converted into the continuous suspension culture of serum-free at last.Through exploration repeatedly repeatedly; Successful domestication express the Chinese hamster ovary celI strain of rhIL-12; Make its CHO-S-SFM II serum-free medium that is fit to U.S. GIBCO company fully, adopt the bright 2L zooblast of German shellfish bio-reactor to carry out continous pouring and cultivate, through concentration monitoring continuously to various nutrient concentrations and meta-bolites in the culturing process; The culture condition parameter is continued to optimize; The concentration of culture supernatant liquid target protein obviously improves, about 25 days of each cultured continuously, and cell density is up to 1 * 10 ⁷/ ml can gather in the crops about 20L culture supernatant liquid altogether, and Chinese hamster ovary celI can be expressed rhIL-12 by stability and high efficiency, and mean concns is 6 μ g/ml.

Embodiment 8

The purifying of IL-12

The cell culture supernatant that will contain IL-12 carries out ultrafiltration or dilution, cationic exchange coloum on the adjustment salt ion intensity, and (20mM PBS, 1M NaCI pH6.0) carry out stepwise elution, and substep is collected separated portion with buffer; (the NH that will contain IL-12 component 4M ₄) ₂SO ₄(pH8.0) deposition goes deposition to get drainage column on the supernatant, with buffer (20mM Tris-Cl, 2M (NH ₄) ₂SO ₄, pH8.5) carrying out stepwise elution, substep is collected separated portion; To contain and go up anion-exchange column after the IL-12 component is suitably diluted the adjustment ionic strength, (20mM Tris-Cl, 1M NaCl pH8.5) carry out stepwise elution, and substep is collected separated portion with buffer; To contain molecular sieve chromatography on the IL-12 component; (120mM NaCl, 20mM PB pH7.0) carry out wash-out, and substep is collected separated portion, and through SDS-PAGE, methods such as HPLC detect purity and reach more than 98% with buffer.

Embodiment 9

The quality control of rhIL-12

1.rhIL-12 molecular-weight determination

(1) SDS-PAGE determining molecular weight

SDS-PAGE shows that the relative molecular weight of IL-12 is 60-80kD (Fig. 9).

(2) mass spectrum MALDI-TOF detection molecules amount

Purifying obtains to such an extent that use after the desalination of IL-12 sample 50% acetonitrile to be made into the protein soln that concentration is 0.05～0.5g/L, gets 5 μ L solution and mixes with 5 μ L matrix (SA), gets 0.5～1 μ L mixed solution point on probe, after the specimen preparation appearance is drained on kind.Testing tool model: AutoFlex MALDI-TOF-MS; Testing conditions: N2 laser source, wavelength 337nm; Detection mode: linear mode (flight pipe range 1.22M, acceleration voltage 20KV).The determining molecular weight result is: 60013.338Da (Figure 10).Bibliographical information P35 molecular weight subunit is 22513Da; The P40 molecular weight subunit is 34699Da; Add the molecular weight of sugar chain part, MALDI-TOF-MS determining molecular weight 60013.34Da is consistent with theory.

2.rhIL-12 Western Blot detect

Analyze Recombinant Protein Expression with immunoblotting Western Blot.With after specific antibody combines, antibody combines with the enzyme labelled antibody specificity this genealogy of law for test product, through the colour developing of zymetology reaction, the antigen-specific of trial-product is checked.The Western Blot result of each batch rhIL-12 stoste is consistent, and (Figure 11) is positive.

3.rhIL-12 mass spectrum peptide figure measure

Behind trypsin hydrolyzing, 0 adopts mass spectrum (MS) method to measure, and adopting the N2 laser source of wavelength 337nm to detect ionic type during mensuration is positive ion; Detection mode is reflection mode (flight pipe range 2.7M; Acceleration voltage 20KV, reflected voltage 23KV) detection matrix is CCA, result such as Figure 12.

According to the theoretical hydrolysis of theoretical trypsinase site, to peptide quality enzyme spectrum analysis, the peptide section (result such as table 2) that mass spectrum peptide figure and theoretical sequence are complementary meets theoretical expected value.

The peptide section that table 2 mass spectrum peptide figure and theoretical sequence are complementary

4.rhIL-12 activity detect

The rhIL-12 biological activity assay adopts PBMC IFN-γ to induce method

(1) dilution of NIBSC standard substance (WHO international standard substance) and purifying rhIL-12: standard substance and to be diluted to following concentration: 8ng/mL, 4ng/mL, 2ng/ml, 1ng/mL, 0.5ng/mL, 0.25ng/mL, 0.125ng/mL, 0.0625ng/mL through accurate quantitative purifying rhIL-12 with the RMPI1640 basic culture solution for use.

(2) separate the PBMC cell: venous blood samples 5mL, anticoagulant heparin, Hank liquid equivalent mixing; Get the 5mlFicoll lymphocyte separation medium and place the 15mL graduated centrifuge tube, the blood that dilutes slowly is superimposed on the parting liquid along tube wall, form interface clearly; Put in the horizontal centrifuge, the centrifugal 20min of 2000r/min is placed in another graduated centrifuge tube with the direct sucking-off mononuclearcell of dropper; Add the above Hank liquid of 4 times of amounts, abundant mixing, the centrifugal 10min of 1000r/min; The supernatant that inclines washes twice with Hank liquid again, with the RMPI1640 basic culture solution preparation cell suspension that contains 10% inactivated fetal bovine serum; Counting cells is expected the blue cell viability that detects with platform simultaneously, requires Xi Baohuoli>More than 95%, every hole adds 100uL cell suspension and corresponding weaker concn standard substance and purifying rhIL-12 to 96 well culture plate, and the blank group adds the RMPI1640 basic culture solution, and positive controls adds anti-people CD ₃, final concentration (0.2ug/mL), each extent of dilution are done two multiple holes.

(3) 37 ℃, 5%CO ₂, cultivated 48 hours

(4) the every hole 50ul of collecting cell culture supernatant liquid, ELISA after the centrifugal treating (pressing the test kit specification sheets operation of BD company) measures IFN-γ content.

(5) result calculates: measure each hole absorbancy, Ascent software version2.6 software curve quantitative Analysis IFN-γ content with MULTISKAN MK3 ELIASA (Thermo company) 450nm.Contrast with the corresponding extent of dilution IFN-of purifying rhIL-12 γ generation with each extent of dilution stimulated in vitro PBMCs IFN-γ generation of standard substance.(t check: P>0.05, two sample room there was no significant difference).Or with IFN-γ content sample concentration is mapped, calculate the ED50 (being that IFN-γ content is the sample concentration of a half of peak concentration) of each laboratory sample, by formula: testing sample tires=and standard substance tire * (ED50 of the ED50/ laboratory sample of standard substance).The ratio work of purified rhIL-12 is 6.4 * 10 ⁶More than the IU/mg.

The P35 subunit coding gene sequence of the rhIL-12 (hIL-12) that appendix 1a.GenBank announces

NM_000882.Reports Homo sapiens inte...[gi：24430218]

LOCUS NM_000882 1444bp mRNA linear PRI03-DEC-2006

DEFINITION Homo sapiens interleukin 12A(natural killer cell stimulatory

factor1，cytotoxic lymphocyte maturation factor1，p35)(IL12A)，

mRNA.

ACCESSION NM_000882

VERSION NM_000882.2GI：24430218

KEYWORDS.

SOURCE Homo sapiens(human)

ORGANISM Homo sapiens

Eukaryota；Metazoa；Chordata；Craniata；Vertebrata；Euteleostomi；

Mammalia；Eutheria；Euarchontoglires；Primates；Haplorrhini；

Catarrhini；Hominidae；Homo.

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COMMENT REVIEWED REFSEQ：This record has been curated by NCBI staff.

The

reference sequence was derived from M65291.1，AW472810.1and

BG702253.1.

OnOct30，2002this sequence version replaced gi:4504638.

Summary：This gene encodes a subunit of a cytokine that acts on T

and natural killer cells，and has a broad array of biological

activities.The cytokine is a disulfide-linked heterodimer composed

of the35-kD subunit encoded by this gene，and a40-kD subunit that

is a member ofthe cytokine receptor family.This cytokine is

required for the T-cell-independent induction of interferon

(IFN)-gamma，and is important for the differentiation of both Th1

and Th2cells.The responses of lymphocytes to this cytokine are

mediated by the activator of transcription protein STAT4.Nitric

oxide synthase2A(NOS2A/NOS2)is found to be required for the

signaling process of this cytokine in innate immunity.

Publication Note：This RefSeq record includes a subset of the

publications that are available for this gene.Please see the

Entrez Gene record to access additional publications.

COMPLETENESS：complete on the3′end.

FEATURES Location/Qualifiers

source 1..1444

/organism＝＂Homo sapiens＂

/mol_type＝＂mRNA＂

/db_xref＝＂taxon:9606＂

/chromosome＝＂3＂

/map＝＂3p12-q13.2＂

gene 1..1444

/gene＝＂IL12A＂

/note＝＂interleukin12A(natural killer cellstimulatory

factor1，cytotoxic lymphocyte maturation factor1，p35)；

synonyms：CLMF，NFSK，NKSF1，IL-12A＂

/db_xref＝＂GeneID：3592＂

/db_xref＝＂HGNC：5969＂

/db_xref＝＂HPRD：01193＂

/db_xref＝＂MIM：161560＂

CDS 216..977

/gene＝＂IL12A＂

/note＝＂natural killer cell stimulatoryfactor1，35kD

subunit；cytotoxic lymphocyte maturationfactor1，p35；

interleukin12，p35；IL-12，subunitp35；NF cell

stimulatory factor chain1；interleukin-12alpha chain

precursor＂

/codon_start＝1

/product＝＂interleukin12A precursor＂

/protein_id＝＂NP_000873.2＂

/db_xref＝＂GI：24430219＂

/db_xref＝＂CCDS：CCDS3187.1＂

/db_xref＝＂GeneID：3592＂

/db_xref＝＂HGNC：5969＂

/db_xref＝＂HPRD：01193＂

/db_xref＝＂MIM：161560＂

/translation＝＂MWPPGSASQPPPSPAAATGLHPAARPVSLQCRLSMCPARSLLLV

ATLVLLDHLSLARNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPC

TSEE

IDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCL

SS

IYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQ

KSSLE

EPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS＂

sig_peptide 216..383

/gene＝＂IL12A＂

mat_peptide 384..974

/gene＝＂IL12A＂

/product＝＂interleukin12A＂

STS 915..1340

/gene＝＂IL12A＂

/standard_name＝＂PMC105864P1＂

/db_xref＝＂UniSTS：270118＂

STS 915..1197

/gene＝＂IL12A＂

/standard_name＝＂GDB：252062＂

/db_xref＝＂UniSTS：156323＂

STS 972..1103

/gene＝＂IL12A＂

/standard_name＝＂RH17729＂

/db_xref＝＂UniSTS:66784＂

STS 989..1336

/gene＝＂IL12A＂

/standard_name＝＂SHGC-12751＂

/db_xref＝＂UniSTS：32985＂

polyA_signal 1414..1419

/gene＝＂IL12A＂

polyA_site 1437

/gene＝＂IL12A＂

/experiment＝＂experimental evidence，no additional details

recorded＂

ORIGIN

1 tttcattttg ggccgagctg gaggcggcgg ggccgtcccg gaacggctgc ggccgggcac

61 cccgggagtt aatccgaaag cgccgcaagc cccgcgggcc ggccgcaccg cacgtgtcac

121 cgagaagctg atgtagagag agacacagaa ggagacagaa agcaagagac cagagtcccg

181 ggaaagtcct gccgcgcctc gggacaatta taaaaatgtg gccccctggg tcagcctccc

241 agccaccgcc ctcacctgcc gcggccacag gtctgcatcc agcggctcgc cctgtgtccc

301 tgcagtgccg gctcagcatg tgtccagcgc gcagcctcct ccttgtggct accctggtcc

361 tcctggacca cctcagtttg gccagaaacc tccccgtggc cactccagac ccaggaatgt

421 tcccatgcct tcaccactcc caaaacctgc tgagggccgt cagcaacatg ctccagaagg

481 ccagacaaac tctagaattt tacccttgca cttctgaaga gattgatcat gaagatatca

541 caaaagataa aaccagcacagtggaggcct gtttaccatt ggaattaacc aagaatgaga

601 gttgcctaaa ttccagagag acctctttca taactaatgg gagttgcctg gcctccagaa

661 agacctcttt tatgatggcc ctgtgccttagtagtattta tgaagacttg aagatgtacc

721 aggtggagtt caagaccatg aatgcaaagc ttctgatgga tcctaagagg cagatctttc

781 tagatcaaaa catgctggca gttattgatg agctgatgca ggccctgaat ttcaacagtg

841 agactgtgcc acaaaaatcc tcccttgaag aaccggattt ttataaaact aaaatcaagc

901 tctgcatact tcttcatgct ttcagaattc gggcagtgac tattgataga gtgatgagct

961 atctgaatgc ttcctaaaaa gcgaggtccc tccaaaccgt tgtcattttt ataaaacttt

1021 gaaatgagga aactttgata ggatgtggat taagaactag ggagggggaa agaaggatgg

1081 gactattaca tccacatgat acctctgatc aagtattttt gacatttact gtggataaat

1141 tgtttttaag ttttcatgaa tgaattgcta agaagggaaa atatccatcc tgaaggtgtt

1201 tttcattcac tttaatagaa gggcaaatat ttataagcta tttctgtacc aaagtgtttg

1261 tggaaacaaa catgtaagca taacttattt taaaatattt atttatataa cttggtaatc

1321 atgaaagcat ctgagctaac ttatatttat ttatgttata tttattaaat tatttatcaa

1381 gtgtatttga aaaatatttt taagtgttct aaaaataaaa gtattgaatt aaagtgaaaa

1441 aaaa

//

The P40 subunit coding gene sequence of the rhIL-12 (hIL-12) that appendix 1bGenBank announces

1：NM_002187.Reports Homo sapiens inte...[gi：24497437]

LOCUS NM_002187 2347bp mRNA linear PRI26-NOV-2006

DEFINITION Homo sapiens interleukin12B(natural killer cellstimulatory

fact2，cytotoxiclymphocyte maturation factor 2，p40)(IL12B)，

mRNA.

ACCESSION NM_002187

VERSION NM_002187.2GI：24497437

KEYWORDS.

SOURCE Homo sapiens(human)

ORGANISM Homo sapiens

Eukaryota；Metazoa；Chordata；Craniata；Vertebrata；Euteleostomi；

Mammalia；Eutheria；Euarchontoglires；Primates；Haplorrhini；

Catarrhini；Hominidae；Homo.

REFERENCE 1 (bases1to2347)

AUTHORS Vujisic，S.，Lepej，S.Z.，Emedi，I.，Bauman，R.，Remenar，A.and

Tiljak，M.K.

TITLE Ovarian follicular concentration of IL-12，IL-15，IL-18and p40

subunit of IL-12and IL-23

JOURNAL Hum.Reprod.21(10)，2650-2655(2006)

PUBMED 16772281

REMARK GeneRIF：Increased concentrations of p40subunit of IL-12/IL-23in

follicles containing oocytes suggest an important role of this

cytokine in reproduction.

REFERENCE 2 (bases1to2347)

AUTHORS Suneetha，P.V.，Goyal，A.，Hissar，S.S.and Sarin，S.K.

TITLE Studies on TAQ1polymorphism in the3′untranslatedregion of

IL-12P40gene in HCV patients infected predominantlywith genotype

3

JOURNAL J.Med.Virol.78(8)，1055-1060(2006)

PUBMED 16789008

REMARK GeneRIF：The association of IL-12p40A1188C polymorphism with the

outcome of HCV infection is reported.

REFERENCE 3 (bases1to2347)

AUTHORS Yanagihori，H.，Oyama，N.，Nakamura，K.，Mizuki，N.，Oguma，K.and

Kaneko，F.

TITLE Role of IL-12B promoter polymorphism inAdamantiades-Behcet′s

disease susceptibility：Aninvolvement of Th1immunoreactivity

against Streptococcus Sanguinis antigen

JOURNAL J.Invest.Dermatol.126(7)，1534-1540(2006)

PUBMED 16514412

REMARK GeneRIF：Our results provide evidence for anti-bacterial host

response toward Th1-immunity mediated by IL-12B inpatients with

Adamantiades-Behcet′s disease(ABD)，and the possible insight into

the genetic susceptibilitythatis independent of HLA background.

REFERENCE 4 (bases1to2347)

AUTHORS Yoshikawa，H.，Sato，K.，Edahiro，S.，Furukawa，Y.，Maruta，T.，

Iwasa，K.，Watanabe，H.，Takaoka，S.，Suzuki，Y.，Takamori，M.and

Yamada，M.

TITLE Elevation of IL-12p40and its antibody in myasthenia gravis with

thymoma

JOURNAL J.Neuroimmunol.175(1-2)，169-175(2006)

PUBMED 16574246

REMARK GeneRIF：Among cytokines affecting T helper type1(Th1)-Th2

balance，the levels of IL-12p40and its autoantibody are

specifically elevated in patients with myasthenia gravis

complicated by thymoma.

REFERENCE 5 (bases 1 to2347)

AUTHORS Silswal，.N.Singh，A.K.，Aruna，B.，Mukhopadhyay，S.，Ghosh，S.and

Ehtesham，N.Z.

TITLE Human resistin stimulates the pro-inflammatory cytokines TNF-alphaa

and IL-12 in macrophages by NF-kappaB-dependent pathway

JOURNAL Biochem.Biophys.Res.Commun.334(4)，1092-1101(2005)

PUBMED 16039994

REMARK GeneRIF：The pro-infiammatory nature of hResistin was further

evident from the abillty of this protein to induce the nuclear

translocation of NF-kappaB tramscroption factor as seen from

electrophoretic mobility shift assays.

REFERENC 6 (bases 1 to 2347)

AUTHORS Ling.P.，Gately，Gubler，U.，Stern，A.S.，Lin，P.，Hollfelder，K.，

Su，C.，Pan，Y.C.and Hakimi，J.

TITLE Human IL-12p40 homodimer binds to the IL-12receptor but does not

mediate biologic activity

JOURNAL J.Immunol. 154(1)，116-127(1995)

PUBMED 7527811

REFERENCE 7 (bades 1 to 2347)

AUTHORS Aragane，y.，Riemann，H.,Bhardwaj,R.S.Schwarz，A.,Sawada，Y.，

Yamada，H.，Luger，T.A.，Kubln，M.，Trinchierl，g.and Schwarz，T.

TITLE IL-12 is expressed and released by human keratinocytes and

epidermoid carcinoma cell lines

JOURNAL J.Immunol .153(12)，5366-5372(1944)

PUBMED 7527439

REFERENCE 8 (bases 1 to 2347)

AUTHORS Sieburth，D.，Jabs，E.W.，Warrington，.J.A.，Li，X.，Lasota，J.，

LaForgia，S.，Kelleher，K.，Huebmer，K.，Wasmuth，.J.J.and Wolf，S.F.，

TITLE Assignment of genes encoding a unique cytokine (IL12)composed of

two unrelated subunets to chromosomes3 and 5

JOURNAL Genomics 14(1，59-62(1992)

PUBMED 1358798

REFERENCE 9 (bases 1 to 2347)

AUTHORS Gubler，U.，Chua，A.O.，Schoenhaut，D.S.，Dwyer，C.M.，McComas，W.，

Motyka，R.，Nabavi，N.，Wolitzky，A.G.，Quinn，P.M.，Familletti，P.C.et

al.

TITLE Coexpression of two distinct genes is required to generate secreted

bioactive cytotoxic lymphocyte maturation factor

JOURNAL Proc.Natl.Acad.Sci.U.S.A.88(10)，4143-4147(1991)

PUBMED 1674604

REFERENCE 10(bases1to2347)

AUTHORS Wolf，S.F.，Temple，P.A.，Kobayashi，M.，Young，D.，Dicig，M.，Lowe，L.，

Dzialo，R.，Fitz，L.，Ferenz，C.，Hewick，R.M.et al.

TITLE Cloning of cDNA for natural killer cell stimulatory factor，a

heterodimeric cytokine with multiple biologic effects on T and

natural killer cells

JOURNAL J.Immunol.146(9)，3074-3081(1991)

PUBMED 1673147

COMMENT REVIEWED REFSEQ：This record has been curated by NCBI staff.

The

reference sequence was derived from M65290.1and M65272.1.

On Nov3，2002this sequence version replaced gi：4504640.

Summary：This gene encodes a subunit of interleukin12，a cytokine

that acts on T andnatural killer cells，and has a broad array of

biological activities.Interleukin12is a disulfide-linked

heterodimer composed ofthe40kD cytokine receptor like subunit

encoded by this gene，and a35kD subunit encoded by IL12A.This

cytokine is expressed by activated macrophages that serve as an

essential inducer of Th1cells development.This cytokine has been

found to be important for sustaining a sufficient number of

memory/effector Th1cells to mediate long-term protection to an

intracellular pathogen.Overexpression of this gene was observed in

the central nervous system of patients with multiple sclerosis

(MS)，suggesting a role of this cytokine inthe pathogenesis of the

disease.The promoter polymorphism of this gene has been reported

to be associated with the severityof atopic and non-atopic asthma

in children.

Publication Note：This RefSeq record includes a subset of the

publications that are available for this gene.Please see the

Entrez Gene record to access additional publications.

FEATURES Location/Qualifiers

source 1..2347

/organism＝＂Homo sapiens＂

/mol_type＝＂mRNA＂

/db_xref＝＂taxon：9606＂

/chromosome＝＂5＂

/map＝＂5q31.1-q33.1＂

gene 1..2347

/gene＝＂IL12B＂

/note＝＂interleukin12B(natural killer cellstimulatory

factor2，cytotoxic lymphocyte maturationfactor2，p40)；

synonyms：CLMF，NKSF，CLMF2，NKSF2，IL-12B＂

/db_xref＝＂GeneID：3593＂

/db_xref＝＂HGNC：5970＂

/db_xref＝＂HPRD：01194＂

/db_xref＝＂MIM：161561＂

CDS 43..1029

/gene＝＂IL12B＂

/go_component＝＂extracellular space[pmid1673147]；

membrane＂

/go_function＝＂cytokine activity；

hematopoietin/interferon-class(D200-domain)cytokine

receptor activity；interleukin-12receptor binding[pmid

1674604]；signal transducer activity[pmid9789052]＂

/go_process＝＂antimicrobialhumoral response(sensu

Vertebrata)[pmid10320373]；interferon-gamma biosynthesis

[pmid1673147]；naturalkiller cellactivation[pmid

1673147]；positive regulation of activated T cell

proliferation[pmid1674604]；positive regulation of

interferon-gamma biosynthesis[pmid1673147]；regulation

of cytokine biosynthesis[pmid1673147]；T-helper cell

differentiation[pmid1673147]＂

/note＝＂natural killcr cell stimulatory factor-2；cytotoxic

lymphocyte maturation factor2，p40；interleukin12，p40；

natural killer cell stimulatory factor，40kD subunit；

interleukin-12beta chain；IL12，subunit p40＂

/codon_start＝1

/product＝＂interleukin12B precursor＂

/protein_id＝＂NP_002178.2＂

/db_xref＝＂GI：24497438＂

/db_xref＝＂CCDS：CCDS4346.1＂

/db_xref＝＂GeneID：3593＂

/db_xref＝＂HGNC：5970＂

/db_xref＝＂HPRD：01194＂

/db_xref＝＂MIM：161561＂

/translation＝＂MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEM

VVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSH

SLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLT

FSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIE

VMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWST

PHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSW

SEWASVPCS＂

sig_peptide 43..108

/gene＝＂IL12B＂

mat_peptide 109..1026

/gene＝＂IL12B＂

/product＝＂interleukin12B＂

STS 317..471

/gene＝＂IL12B＂

/standard_name＝＂PMC321570P1＂

/db_xref＝＂UniSTS：273112＂

STS 1053..1272

/gene＝＂IL12B＂

/standard_name＝＂SGC35786＂

/db_xref＝＂UniSTS：51991＂

STS 2015..2195

/gene＝＂IL12B＂

/standard_name＝＂RH17728＂

/db_xref＝＂UniSTS：24645＂

STS 2116..2324

/gene＝＂IL12B＂

/standard_name＝＂G10637＂

/db_xref＝＂UniSTS：60436＂

ORIGIN

1 ctgtttcagg gccattggac tctccgtcct gcccagagca agatgtgtca ccagcagttg

61 gtcatctctt ggttttccct ggtttttctg gcatctcccc tcgtggccat atgggaactg

121 aagaaagatg tttatgtcgtagaattggat tggtatccgg atgcccctgg agaaatggtg

181 gtcctcacct gtgacacccc tgaagaagat ggtatcacct ggaccttgga ccagagcagt

241 gaggtcttag gctctggcaa aaccctgacc atccaagtca aagagtttgg agatgctggc

301 cagtacacct gtcacaaagg aggcgaggtt ctaagccatt cgctcctgct gcttcacaaa

361 aaggaagatg gaatttggtc cactgatatt ttaaaggacc agaaagaacc caaaaataag

421 acctttctaa gatgcgaggc caagaattat tctggacgtt tcacctgctg gtggctgacg

481 acaatcagta ctgatttgac attcagtgtc aaaagcagca gaggctcttc tgacccccaa

541 ggggtgacgt gcggagctgc tacactctct gcagagagag tcagagggga caacaaggag

601 tatgagtact cagtggagtg ccaggaggac agtgcctgcc cagctgctga ggagagtctg

661 cccattgagg tcatggtgga tgccgttcac aagctcaagt atgaaaacta caccagcagc

721 ttcttcatca gggacatcat caaacctgac ccacccaaga acttgcagct gaagccatta

781 aagaattctc ggcaggtgga ggtcagctgg gagtaccctg acacctggag tactccacat

841 tcctacttct ccctgacatt ctgcgttcag gtccagggca agagcaagag agaaaagaaa

901 gatagagtct tcacggacaa gacctcagcc acggtcatct gccgcaaaaa tgccagcatt

961 agcgtgcggg cccaggaccg ctactatagc tcatcttgga gcgaatgggc atctgtgccc

1021 tgcagttagg ttctgatcca ggatgaaaat ttggaggaaa agtggaagat attaagcaaa

1081 atgtttaaag acacaacgga atagacccaa aaagataatt tctatctgat ttgctttaaa

1141 acgttttttt aggatcacaa tgatatcttt gctgtatttg tatagttaga tgctaaatgc

1201 tcattgaaac aatcagctaa tttatgtata gattttccag ctctcaagtt gccatgggcc

1261 ttcatgctat ttaaatattt aagtaattta tgtatttatt agtatattac tgttatttaa

1321 cgtttgtctg ccaggatgta tggaatgttt catactctta tgacctgatc catcaggatc

1381 agtccctatt atgcaaaatg tgaatttaat tttatttgta ctgacaactt ttcaagcaag

1441 gctgcaagta catcagtttt atgacaatca ggaagaatgc agtgttctga taccagtgcc

1501 atcatacact tgtgatggat gggaacgcaa gagatactta catggaaacc tgacaatgca

1561 aacctgttga gaagatccag gagaacaaga tgctagttcc catgtctgtg aagacttcct

1621 ggagatggtg ttgataaagc aatttagggc cacttacact tctaagcaag tttaatcttt

1681 ggatgcctga attttaaaag ggctagaaaa aaatgattga ccagcctggg aaacataaca

1741 agaccccgtc tctacaaaaa aaatttaaaa ttagccaggc gtggtggctc atgcttgtgg

1801 tcccagctgt tcaggaggat gaggcaggag gatctcttga gcccaggagg tcaaggctat

1861 ggtgagccgt gattgtgcca ctgcatacca gcctaggtga cagaatgaga ccctgtctca

1921 aaaaaaaaaa tgattgaaat taaaattcag ctttagcttc catggcagtc ctcaccccca

1981 cctctctaaa agacacagga ggatgacaca gaaacaccgt aagtgtctgg aaggcaaaaa

2041 gatcttaaga ttcaagagag aggacaagta gttatggcta aggacatgaa attgtcagaa

2101 tggcaggtgg cttcttaaca gccctgtgag aagcagacag atgcaaagaa aatctggaat

2161 ccctttctca ttagcatgaa tgaacctgat acacaattat gaccagaaaa tatggctcca

2221 tgaaggtgct acttttaagt aatgtatgtg cgctctgtaa agtgattaca tttgtttcct

2281 gtttgtttat ttatttattt atttttgcat tctgaggctg aactaataaa aactcttctt

2341 tgtaatc

//

Claims (4)

1. the expression vector of a rhIL-12 is characterized in that:

(1) this expression vector comprises two the p35 subunits of people IL-12 and the transcript of a p40 subunit;

(2) p35 subunit, p40 subunit are respectively by separately promotor CMV control; (3) promotor of p35 subunit has designed the enhancer element of SV40 before, between promotor and p35 subunit sequence, has introduced the Intron element again; Its method is: extract total RNA that test kit extracts people's cell with RNeasy, and as the template of reverse transcription PCR, carry out single stage method RT-PCR amplification respectively, obtaining length is the p35 gene product of 684bp and the p40 gene product of length 1002bp;

(4) the IRES element of introducing regulation and control neo genetic expression between p40 subunit and the resistance screening mark neo gene; Its method is: with the carrier pIRESneo that carries Neo resistance screening mark; The PCR product of p40 gene is inserted in the pIRESneo carrier with BamHI single endonuclease digestion rear clone, and screening obtains recombinant plasmid and the called after pP40IRESneo that forward inserts;

(5) two p35 subunit expression units in series are in same carrier; Its method is: 1) carrier pDouble carries double expression boxes; The PCR product of p35 gene is inserted in the pDouble carrier with SalI single endonuclease digestion rear clone, and screening obtains recombinant plasmid and the called after pDouble-P35 that forward inserts; 2), the expression cassette that carries whole P40-IRES-Neo is transferred to the KpnI single endonuclease digestion and mends in the pDouble-P35 carrier after putting down the recombinant plasmid called after pP75 of acquisition with NruI+XhoI double digestion carrier pP40IRESneo;

(6) two p35 subunits are series at before the p40 subunit; Its method is: 1) with NruI+KpnI double digestion carrier pDouble-P35, reclaim P35 subunit expression box 3285bp, and mend flat through the T4 archaeal dna polymerase; 2) with NruI single endonuclease digestion carrier pP75, carry out dephosphorylation 9416bp with the CIP enzyme; 3) connect 1 and 2, filter out positive colony p2P35-1P40 behind the transformed into escherichia coli;

Described expression vector is recombinant plasmid recombinant plasmid p2P35-1P40;

Wherein: p35 subunit amplimer and p40 subunit amplimer primer sequence are:

P35 forward primer sequence is: underscore is represented the SalI restriction enzyme site:

5’-TGG GTCGACGCCACCATGTGTCCAGCGCGCAGCCTC-3’

P35 reverse primer sequence is: underscore is represented the SalI restriction enzyme site:

5’-TGG GTCGACTCAGGAAGCATTCAGATAGC-3’

P40 forward primer sequence is: underscore is represented the BamHI restriction enzyme site:

5’-CG GGATCCATGTGTCACCAGCAGTTG-3’

P35 reverse primer sequence is: underscore is represented the BamHI restriction enzyme site:

5’-CG GGATCCTAACTGCAGGGCACAGATG-3’。

2. expression vector as claimed in claim 1 is characterized in that: the expression modular construction of carrier is CMV promotor-p35 subunit-CMV promotor-p35 subunit-CMV promotor-p40 subunit.

3. expression vector as claimed in claim 2 is characterized in that: the expression modular construction of carrier is following nucleotide sequence: SV40 Enhancer-pCMV-Intron-p35-SV40Enhancer-pCMV-Intron-p35-pC MV-p40-IRES-neo.

4. one kind efficiently expresses people IL-12 eucaryotic cell strain, it is characterized in that: in the CHO mammal cell line, prepare, said cell contains the described expression vector of claim 1, and perhaps said cell has been used the described nucleotide sequence transfection of claim 3.

Images